ISO 23196:2022
(Main)Water quality — Calculation of biological equivalence (BEQ) concentrations
Water quality — Calculation of biological equivalence (BEQ) concentrations
This document specifies the derivation of biological equivalence (BEQ) concentrations for results of in vitro bioassays which are based on measuring effects on a biological process such as enzyme induction or cellular growth. The concept described here can be used for any biological assay after the proof of its applicability. To derive BEQ concentrations, the effect on a biological process caused by a sample – i.e. the activity of the sample – is expressed in terms of a concentration of a reference compound which results in an equivalent effect on the process. The term "sample" used in this document addresses environmental samples as well as defined mixtures and pure compounds used as test item in a bioassay. BEQ concentrations can be derived for environmental water samples, extracts of environmental water samples including tap water or solutions of pure chemicals or mixtures of chemicals.
Qualité de l’eau — Calcul des concentrations en équivalent biologique (BEQ)
Le présent document spécifie la détermination de concentrations en équivalent biologique (BEQ) à partir de résultats de bioessais in vitro qui sont fondés sur la mesure des effets produits sur un processus biologique tel que l’induction d’enzymes ou la croissance cellulaire. Le concept décrit ici peut être utilisé pour n’importe quel bioessai du moment que son applicabilité a été démontrée. Pour déterminer les concentrations BEQ, l’effet produit par un échantillon sur un processus biologique, c’est-à-dire l’activité de l’échantillon, est exprimé sous la forme de la concentration d’un composé de référence à laquelle un effet équivalent est obtenu sur le processus. Le terme «échantillon» utilisé dans le présent document renvoie à des échantillons environnementaux ainsi qu’à des mélanges déterminés et à des composés purs utilisés comme objets de mesure dans le cadre d’un bioessai. Les concentrations en équivalent biologique peuvent être déterminées pour des échantillons d’eau environnementaux, des extraits d’échantillons d’eau environnementaux, y compris d’eau du robinet, ou des solutions de substances chimiques pures ou de mélanges de substances chimiques.
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INTERNATIONAL ISO
STANDARD 23196
First edition
2022-02
Water quality — Calculation of
biological equivalence (BEQ)
concentrations
Reference number
ISO 23196:2022(E)
© ISO 2022
---------------------- Page: 1 ----------------------
ISO 23196:2022(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2022
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
© ISO 2022 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 23196:2022(E)
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Procedure .5
5.1 General . 5
5.2 Procedure for the calculation of biological equivalence (BEQ) concentrations . 5
5.2.1 Assessment of the suitability of the experimental data for the calculation
of a biological equivalence (BEQ) concentration . 5
5.2.2 Fitting of concentration-effect data for the reference compound . 8
5.2.3 Calculation of quality measures for the fit . 9
5.2.4 Normalization of data from the reference compound and samples . 10
5.2.5 Calculation of the x %-effect level of the reference concentration-effect
relationship and the respective RC -value . 11
x
5.2.6 Assessment of the validity of the experimental data for the calculation of a
biological equivalence (BEQ) concentration .12
5.2.7 Calculation of the concentration factor of the sample at x %-effect level by
linear interpolation .12
5.2.8 Calculation of the biological equivalence (BEQ) concentration .13
6 Validity criteria .14
7 Test report .14
2
Annex A (informative) Illustration of χ statistics using data from Table 1 .15
Bibliography .18
iii
© ISO 2022 – All rights reserved
---------------------- Page: 3 ----------------------
ISO 23196:2022(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to
the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5,
Biological methods.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv
© ISO 2022 – All rights reserved
---------------------- Page: 4 ----------------------
INTERNATIONAL STANDARD ISO 23196:2022(E)
Water quality — Calculation of biological equivalence
(BEQ) concentrations
1 Scope
This document specifies the derivation of biological equivalence (BEQ) concentrations for results of in
vitro bioassays which are based on measuring effects on a biological process such as enzyme induction
or cellular growth. The concept described here can be used for any biological assay after the proof of its
applicability.
To derive BEQ concentrations, the effect on a biological process caused by a sample – i.e. the activity
of the sample – is expressed in terms of a concentration of a reference compound which results in an
equivalent effect on the process. The term "sample" used in this document addresses environmental
samples as well as defined mixtures and pure compounds used as test item in a bioassay. BEQ
concentrations can be derived for environmental water samples, extracts of environmental water
samples including tap water or solutions of pure chemicals or mixtures of chemicals.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
biological equivalence concentration
BEQ concentration
concentration of a reference compound (3.6) that causes the same effect as the effect measured in a
sample, a sample dilution or a solution containing one or more chemicals
3.2
concentration-effect relationship
response to a concentration gradient of an environmental sample or a known substance or mixture of
substances which is described by pre-determined diagnostic indicators
[SOURCE: ISO 6107:2021, 3.127, modified — the term "environmental sample" added; Note 1 to entry
has been deleted.]
3.3
concentration factor
CF
ratio of the actual concentration of the sample compared to the original sample taking all enrichment
and dilution steps of the sample into account
1
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---------------------- Page: 5 ----------------------
ISO 23196:2022(E)
3.4
limit of quantification
LOQ
lowest value of a determinant that can be determined with an acceptable level of accuracy and precision
[SOURCE: ISO 15839:2003, 3.18]
3.5
negative control material
well characterized material and/or substance that, when evaluated by a specific test method,
demonstrates the suitability of the test system to yield a reproducible, appropriately negative,
non-reactive or minimal response in the test system
Note 1 to entry: In practice, negative controls include blanks, vehicles/solvents and reference materials.
[SOURCE: ISO 7405:2018, 3.4, modified — Note 1 to entry has been deleted.]
3.6
reference compound
RC
compound with one or more property values that are sufficiently reproducible and well established to
enable the calibration of the measurement method
Note 1 to entry: For the purpose of this document, a reference compound is any well characterized material and/
or substance that, when tested by the procedure described, demonstrates the suitability of the procedure to yield
a reproducible, predictable positive response.
[SOURCE: ISO 7405:2018, 3.5, modified — "compound" replaces "material"; "the calibration of the
measurement method" replaces "use of the material or substance for the calibration of an apparatus,
the assessment of a measurement method or for the assignment of values to materials". Note 1 was
modified to cover only a reference compound resulting in a positive response – otherwise the proposed
concept is not applicable.]
3.7
x %-effect concentration
EC
x
concentration at which a specific effect is detected; x is the percentage (e.g. 10, 25, 50) of this effect, e.g.
growth inhibition
[SOURCE: ISO 15952:2018, 3.6]
3.8
x %-effect concentration of a reference compound
RC
x
concentration of a reference compound (3.6) at which a specific effect is detected; x is the percentage
(e.g. 10, 25, 50) of this effect, e.g. growth inhibition
4 Principle
The general idea for the derivation of BEQ concentrations, assessed by the test method, is shown in
Figure 1. The biological activity of a sample is expressed in terms of a concentration of a reference
compound which affects a biological process to the same extent. By this means, BEQ concentrations
allow an indirect quantification of results and a comparison of results obtained by different laboratories.
Furthermore, a robust way to calculate BEQ concentrations is a necessary requirement for a possible
[5],[6],[7],[8]
use of effect-based trigger (EBT) values in regulations .
2
© ISO 2022 – All rights reserved
---------------------- Page: 6 ----------------------
ISO 23196:2022(E)
Key
X concentration of the reference compound, e.g. in ng/l
X1 concentration factor of the sample compared to the original water sample
Y effect in arbitrary units
1 sample diluted
2 sample concentrated
3 biological equivalence (BEQ) concentration
reference compound
sample
NOTE The measured effect of a sample or a sample dilution is extrapolated to a concentration of a reference
compound which induces the bioassay to the same extend as the sample or sample dilution to derive a biological
equivalence (BEQ) concentration, here an example of an agonistic action is shown.
Figure 1 — Basic principle of the derivation of a biological equivalence (BEQ) concentration
Several different methods for the derivation of BEQ concentrations have been published, but not all
methods are applicable to all kinds of concentration-effect relationships. For a detailed discussion about
advantages and disadvantages of the various mathematical approaches, see References [1],[3],[5],[6].
The method described in this document, termed RC -approach, is reported to be a robust method for
x
[11],[12] [13]
the derivation of BEQ concentrations and outlined as well in OECD 455 .
NOTE In the literature, other terminologies might be found for RC , such as PC . PC describes the
x 10 10
concentration of the positive control that induces a 10 % effect-level. In this document, the term “reference
compound” is used instead of “positive control”. Therefore, RC is used instead of PC.
The approach is illustrated in Figure 2. As a first step of the RC -approach, the concentration-effect
x
relationship of the reference compound is modelled to determine the maximum effect of the assay for
the reference compound. Next, the effect levels are normalized to a percentage scale whereby the effect
3
© ISO 2022 – All rights reserved
---------------------- Page: 7 ----------------------
ISO 23196:2022(E)
level of the negative control material is defined as 0 % and the maximum effect level of the reference
compound is defined as 100 %. Then, the concentration of the reference compound that affects the
assay to the x %-effect level is calculated (RC ).
x
Key
X concentration of the reference compound, e.g. in ng/l
X1 concentration factor of the sample compared to the original water sample
Y normalized effect in %
RC concentration of the reference compound at 10 % effect level
10
CF concentration factor of the sample at 10 % effect level with respect to the reference compound
10
reference compound
sample
NOTE Following data normalization, where 0 % equals the effect in the negative control material and 100 %
is the modelled maximum effect of the reference. The sample concentration factor (CF) and reference compound
concentration (RC) required to reach an effect level of x % (here 10 %) are interpolated from the data using
linear interpolation (dashed line). The BEQ concentration is derived by dividing RC by CF .
x x
Figure 2 — Illustration of the RC -approach for the derivation of a biological equivalence (BEQ)
x
concentration
In a further step, the concentration factor of the sample is determined at which the sample affects the
assay to the selected x %-effect level (CF ). As defined in 3.3, the concentration factor describes the ratio
x
of the actual concentration of the sample compared to the original water sample taking all enrichment
and dilution steps of the sample into account. If a sample is, under consideration of a quantitative
process, enriched 1 000-fold by solid phase extraction, the final concentration factor of the sample after
a 100-fold dilution with, for example, growth medium is 10. If this sample is tested in a series of six
1→2 dilutions, the resulting rounded concentration factors are 10, 5, 2,5, 1,25, 0,62, 0,31 and 0,15. The
biological equivalence (BEQ) concentration of the sample is finally given by the ratio RC /CF .
x x
4
© ISO 2022 – All rights reserved
---------------------- Page: 8 ----------------------
ISO 23196:2022(E)
5 Procedure
5.1 General
Usually, concentration-effect relationships of reference compounds and samples (see Clause 1) in
reporter gene assays and proliferation assays are sigmoidal. Depending on the individual shape of
the concentration-effect relationship, a suitable mathematical model has to be selected for fitting. In
[14],[15]
general, a five parametric logistic model can be applied as shown in 5.2.
The overall procedure for the calculation of BEQ concentrations consists of the following steps that are
described in detail in 5.2:
— assessment of the suitability of the experimental data for the calculation of a biological equivalence
concentration (see 5.2.1);
— fitting of concentration-effect data for the reference compound (see 5.2.2);
— calculation of quality measures for the fit (see 5.2.3);
— normalization of data from the reference compound and samples (see 5.2.4);
— calculation of the x %-effect level of the reference compound and the respective RC -value (see
x
5.2.5);
— assessment of the validity of the experimental data for the calculation of a biological equivalence
concentration (see 5.2.6);
— calculation of the concentration factor of the sample at the x %-effect level by linear interpolation
(see 5.2.7);
— calculation of the biological equivalence (BEQ) concentration (see 5.2.8).
5.2 Procedure for the calculation of biological equivalence (BEQ) concentrations
5.2.1 Assessment of the suitability of the experimental data for the calculation of a biological
equivalence (BEQ) concentration
To assess the validity of the experimental data for the calculation of biological equivalence (BEQ)
concentrations (see 5.2.6) by the procedure described in this document, some calculations should
be performed. The experimental data should be assessed prior to this procedure as described below
to evaluate its general suitability. Use only experimental data which fulfil the validity criteria of the
respective standard or guideline for the calculation of biological equivalence (BEQ) concentrations.
Use the following guidance to assess a general suitability of the data:
a) at least two more data points than the number of parameters describing the logistic function of the
curve are required for the concentration-effect relationship of the reference compound, i.e. in case
of the five-parametric logistic function described in 5.2.2 seven data points are required;
b) the upper curve plateau of the sigmoidal concentration-effect relationship of the reference
compound is indicated by the data, i.e. might be estimated by the human eye;
c) the lower curve plateau of the sigmoidal concentration-effect relationship of the reference
compound is indicated by the data, i.e. might be estimated by the human eye;
d) the effect measures of the tested sample concentrations are likely to cross the chosen x %-effect
level.
Figure 3 shows two examples to guide the assessment of the suitability of the experimental data for the
calculation of an equivalence concentration.
5
© ISO 2022 – All rights reserved
---------------------- Page: 9 ----------------------
ISO 23196:2022(E)
Figure 3 a) shows two concentration-effect relationships of the reference compound that are not suitable
for the calculation of biological equivalence (BEQ) concentrations. In case of the upper curve (black
triangles, high response curve), the bottom of the concentration-effect relationship is not defined; in
case of the lower curve (black squares, low response curve), the top (maximum to infinite effect range)
is not defined. In such cases, adjust the concentration range for the reference compound to generate a
complete concentration-effect relationship.
F
...
FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 23196
ISO/TC 147/SC 5
Water quality — Calculation of
Secretariat: DIN
biological equivalence (BEQ)
Voting begins on:
2021-10-08 concentrations
Voting terminates on:
2021-12-03
RECIPIENTS OF THIS DRAFT ARE INVITED TO
SUBMIT, WITH THEIR COMMENTS, NOTIFICATION
OF ANY RELEVANT PATENT RIGHTS OF WHICH
THEY ARE AWARE AND TO PROVIDE SUPPOR TING
DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
Reference number
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
ISO/FDIS 23196:2021(E)
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN-
DARDS TO WHICH REFERENCE MAY BE MADE IN
NATIONAL REGULATIONS. © ISO 2021
---------------------- Page: 1 ----------------------
ISO/FDIS 23196:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
© ISO 2021 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/FDIS 23196:2021(E)
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Procedure .5
5.1 General . 5
5.2 Procedure for the calculation of biological equivalence (BEQ) concentrations . 5
5.2.1 Assessment of the suitability of the experimental data for the calculation
of a biological equivalence (BEQ) concentration . 5
5.2.2 Fitting of concentration-effect data for the reference compound . 8
5.2.3 Calculation of quality measures for the fit . 9
5.2.4 Nomalization of data from the reference compound and samples . 10
5.2.5 Calculation of the x %-effect level of the reference concentration-effect
relationship and the respective RC -value . 11
x
5.2.6 Assessment of the validity of the experimental data for the calculation of a
biological equivalence (BEQ) concentration .12
5.2.7 Calculation of the concentration factor of the sample at x %-effect level by
linear interpolation .12
5.2.8 Calculation of the biological equivalence (BEQ) concentration .13
6 Validity criteria .14
7 Test report .14
2
Annex A (informative) Illustration of χ statistics using data from Table 1 .15
Bibliography .18
iii
© ISO 2021 – All rights reserved
---------------------- Page: 3 ----------------------
ISO/FDIS 23196:2021(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to
the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5,
Biological methods.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv
© ISO 2021 – All rights reserved
---------------------- Page: 4 ----------------------
FINAL DRAFT INTERNATIONAL STANDARD ISO/FDIS 23196:2021(E)
Water quality — Calculation of biological equivalence
(BEQ) concentrations
1 Scope
This document specifies the derivation of biological equivalence (BEQ) concentrations for results of in
vitro bioassays which are based on measuring effects on a biological process such as enzyme induction
or cellular growth. The concept described here can be used for any biological assay after the proof of its
applicability.
To derive BEQ concentrations, the effect on a biological process caused by a sample – i.e. the activity
of the sample – is expressed in terms of a concentration of a reference compound which results in an
equivalent effect on the process. The term "sample" used in this document addresses environmental
samples as well as defined mixtures and pure compounds used as test item in a bioassay. BEQ
concentrations can be derived for environmental water samples, extracts of environmental water
samples including tap water or solutions of pure chemicals or mixtures of chemicals.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
biological equivalence concentration
BEQ concentration
concentration of a reference compound (3.6) that causes the same effect as the effect measured in a
sample, a sample dilution or a solution containing one or more chemicals
3.2
concentration-effect relationship
response to a concentration gradient of an environmental sample or a known substance or mixture of
substances which is described by pre-determined diagnostic indicators
[SOURCE: ISO 6107:2021, 3.127, modified — the term "environmental sample" added; Note 1 to entry
has been deleted.]
3.3
concentration factor
CF
ratio of the actual concentration of the sample compared to the original sample taking all enrichment
and dilution steps of the sample into account
1
© ISO 2021 – All rights reserved
---------------------- Page: 5 ----------------------
ISO/FDIS 23196:2021(E)
3.4
limit of quantification
LOQ
lowest value of a determinant that can be determined with an acceptable level of accuracy and precision
[SOURCE: ISO 15839:2003, 3.18]
3.5
negative control material
well characterized material and/or substance that, when evaluated by a specific test method,
demonstrates the suitability of the test system to yield a reproducible, appropriately negative,
non-reactive or minimal response in the test system
Note 1 to entry: In practice, negative controls include blanks, vehicles/solvents and reference materials (3.5).
[SOURCE: ISO 7405:2018, 3.4, modified — Note 1 to entry has been deleted.]
3.6
reference compound
RC
compound with one or more property values that are sufficiently reproducible and well established to
enable the calibration of the measurement method
Note 1 to entry: For the purpose of this document, a reference compound is any well characterized material and/
or substance that, when tested by the procedure described, demonstrates the suitability of the procedure to yield
a reproducible, predictable positive response.
[SOURCE: ISO 7405:2018, 3.5, modified — "compound" replaces "material"; "the calibration of the
measurement method" replaces "use of the material or substance for the calibration of an apparatus,
the assessment of a measurement method or for the assignment of values to materials". Note 1 was
modified to cover only a reference compound resulting in a positive response – otherwise the proposed
concept is not applicable.]
3.7
x %-effect concentration
EC
x
concentration at which a specific effect is detected; x is the percentage (e.g. 10, 25, 50) of this effect, e.g.
growth inhibition
[SOURCE: ISO 15952:2018, 3.6]
3.8
x %-effect concentration of a reference compound
RC
x
concentration of a reference compound (3.6) at which a specific effect is detected; x is the percentage
(e.g. 10, 25, 50) of this effect, e.g. growth inhibition
4 Principle
The general idea for the derivation of BEQ concentrations, assessed by the test method, is shown in
Figure 1. The biological activity of a sample is expressed in terms of a concentration of a reference
compound which affects a biological process to the same extent. By this means, BEQ concentrations
allow an indirect quantification of results and a comparison of results obtained by different laboratories.
Furthermore, a robust way to calculate BEQ concentrations is a necessary requirement for a possible
[5],[6],[7],[8]
use of effect-based trigger (EBT) values in regulations .
2
© ISO 2021 – All rights reserved
---------------------- Page: 6 ----------------------
ISO/FDIS 23196:2021(E)
Key
X concentration of the reference compound, e.g. in ng/l
X1 concentration factor of the sample compared to the original water sample
Y effect in arbitrary units
1 sample diluted
2 sample concentrated
3 biological equivalence (BEQ) concentration
reference compound
sample
NOTE The measured effect of a sample or a sample dilution is extrapolated to a concentration of a reference
compound which induces the bioassay to the same extend as the sample or sample dilution to derive a biological
equivalence (BEQ) concentration, here an example of an agonistic action is shown.
Figure 1 — Basic principle of the derivation of a biological equivalence (BEQ) concentration
Several different methods for the derivation of BEQ concentrations have been published, but not all
methods are applicable to all kinds of concentration-effect relationships. For a detailed discussion about
advantages and disadvantages of the various mathematical approaches, see References [1],[3],[5],[6].
The method described in this document, termed RC -approach, is reported to be a robust method for
x
[11],[12] [13]
the derivation of BEQ concentrations and outlined as well in OECD 455 .
NOTE In the literature, other terminologies might be found for RC , such as PC . PC describes the
x 10 10
concentration of the positive control that induces a 10 % effect-level. In this document, the term “reference
compound” is used instead of “positive control”. Therefore, RC is used instead of PC.
The approach is illustrated in Figure 2. As a first step of the RC -approach, the concentration-effect
x
relationship of the reference compound is modelled to determine the maximum effect of the assay for
the reference compound. Next, the effect levels are normalized to a percentage scale whereby the effect
3
© ISO 2021 – All rights reserved
---------------------- Page: 7 ----------------------
ISO/FDIS 23196:2021(E)
level of the negative control material is defined as 0 % and the maximum effect level of the reference
compound is defined as 100 %. Then, the concentration of the reference compound that affects the
assay to the x %-effect level is calculated (RC ).
x
Key
X concentration of the reference compound, e.g. in ng/l
X1 concentration factor of the sample compared to the original water sample
Y normalized effect in %
RC concentration of the reference compound at 10 % effect level
10
CF concentration factor of the sample at 10 % effect level with respect to the reference compound
10
reference compound
sample
NOTE Following data normalization, where 0 % equals the effect in the negative control material and 100 %
is the modelled maximum effect of the reference. The sample concentration factor (CF) and reference compound
concentration (RC) required to reach an effect level of x % (here 10 %) are interpolated from the data using
linear interpolation (dashed line). The BEQ concentration is derived by dividing RC by CF .
x x
Figure 2 — Illustration of the RC -approach for the derivation of a biological equivalence (BEQ)
x
concentration
In a further step, the concentration factor of the sample is determined at which the sample affects the
assay to the selected x %-effect level (CF ). As defined in 3.3, the concentration factor describes the ratio
x
of the actual concentration of the sample compared to the original water sample taking all enrichment
and dilution steps of the sample into account. If a sample is, under consideration of a quantitative
process, enriched 1 000-fold by solid phase extraction, the final concentration factor of the sample after
a 100-fold dilution with, for example, growth medium is 10. If this sample is tested in a series of six
1→2 dilutions, the resulting rounded concentration factors are 10, 5, 2,5, 1,25, 0,62, 0,31 and 0,15. The
biological equivalence (BEQ) concentration of the sample is finally given by the ratio RC /CF .
x x
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ISO/FDIS 23196:2021(E)
5 Procedure
5.1 General
Usually, concentration-effect relationships of reference compounds and samples (see Clause 1) in
reporter gene assays and proliferation assays are sigmoidal. Depending on the individual shape of
the concentration-effect relationship, a suitable mathematical model has to be selected for fitting. In
[14],[15]
general, a five parametric logistic model can be applied as shown in 5.2.
The overall procedure for the calculation of BEQ concentrations consists of the following steps that are
described in detail in 5.2:
— assessment of the suitability of the experimental data for the calculation of a biological equivalence
concentration (see 5.2.1);
— fitting of concentration-effect data for the reference compound (see 5.2.2);
— calculation of quality measures for the fit (see 5.2.3);
— normalization of data from the reference compound and samples (see 5.2.4);
— calculation of the x %-effect level of the reference compound and the respective RC -value (see
x
5.2.5);
— assessment of the validity of the experimental data for the calculation of a biological equivalence
concentration (see 5.2.6);
— calculation of the concentration factor of the sample at the x %-effect level by linear interpolation
(see 5.2.7);
— calculation of the biological equivalence (BEQ) concentration (see 5.2.8).
5.2 Procedure for the calculation of biological equivalence (BEQ) concentrations
5.2.1 Assessment of the suitability of the experimental data for the calculation of a biological
equivalence (BEQ) concentration
To assess the validity of the experimental data for the calculation of biological equivalence (BEQ)
concentrations (see 5.2.6) by the procedure described in this document, some calculations should
be performed. The experimental data should be assessed prior to this procedure as described below
to evaluate its general suitability. Use only experimental data which fulfil the validity criteria of the
respective standard or guideline for the calculation of biological equivalence (BEQ) concentrations.
Use the following guidance to assess a general suitability of the data:
a) at least two more data points than the number of parameters describing the logistic function of the
curve are required for the concentration-effect relationship of the reference compound, i.e. in case
of the five-parametric logistic function described in 5.2.2 seven data points are required;
b) the upper curve plateau of the sigmoidal concentration-effect relationship of the reference
compound is indicated by the data, i.e. might be estimated by the human eye;
c) the lower curve plateau of the sigmoidal concentration-effect relationship of the reference
compound is indicated by the data, i.e. might be estimated by the human eye;
d) the effect measures of the tested sample concentrations are likely to cross the chosen x %-effect
level.
Figure 3 shows two examples to guide the assessment of the suitability of the experimental data for the
calculation of an equivalence concentration.
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ISO/FDIS 23196:2021(E)
Figure 3 a) shows two concentration-effect relationships of the reference compound that are not suitable
for the calculation of biological equivalence (BEQ) concentrations. In case of the upper curve (black
triangles, high response curve), the bottom of the concentration-effect relationship is not defined; in
case of the lower curve (black s
...
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